BSA - (Aug/14/2005 )

When do immuno, What do BSA and goat serum target respectively. If we have added BSA for blocking, why need goat serum then.

-xiaoben-

The BSA will block non-specific protein interactions and is traditional blocking method for IHC and IF. The serum added should match the species in which the secondary was made in (ie. use goat serum for goat anti-mouse/rabbit secondaries). Some people prefer not to use it. It will likely block Fc receptor binding and non-specific binding of IgG in your secondaries to your sample - this is because the serum contains "non-immune" IgG up to 10 mg/ml.

It is essential though to add some of your block to your antibody diluent. Otherwise you may get your antibodies binding to the blocking agents!

QUOTE (xiaoben @ Aug 14 2005, 09:28 PM)

When do immuno, What do BSA and goat serum target respectively. If we have added BSA for blocking, why need goat serum then.

-MaximinaNYC-

What do you mean by "It will likely block Fc receptor binding and non-specific binding of IgG in your secondaries to your sample "

In our protocal , goat serum is diluated by Ab dilute buffer which contain BSA.So I'm confused why these two are applied together. Which one will block the non specific binding?

By the way , will the BSA bind to goat serum IgG or goat serum bind to BSA IgG? If the former one exists, goat serum seems of no use.

-xiaoben-

They both will block non-specific binding.

Once again, the goat serum contains IgGs which will potentially block any Fc receptors you have in your tissue, if they aren't there in your tissue or cells then it isn't applicable.

BSA is albumin and acts as a non-specific blocking agent. Goat serum is just whole fraction serum and contains many proteins so will also block non-specific binding but because it has IgG may block specifically FcR binding. In addition, since it is the same species as that which your secondary is made in it will block any sites that your secondary may non-specifically bind to.

BSA will not bind to goat serum or vice versa. I use both all the time with no problems. Sometimes people do not like to use serum though as it contains unspecified proteins.

QUOTE (xiaoben @ Aug 15 2005, 08:04 AM)

What do you mean by "It will likely block Fc receptor binding and non-specific binding of IgG in your secondaries to your sample "

In our protocal , goat serum is diluated by Ab dilute buffer which contain BSA.So I'm confused why these two are applied together. Which one will block the non specific binding?

By the way , will the BSA bind to goat serum IgG or goat serum bind to BSA IgG? If the former one exists, goat serum seems of no use.